To the best of the Applicants' knowledge, no prior art whether patented or not has ever successfully exploited the optical transmittance of unaltered whole blood to achieve an accurate measurement of the total hemoglobin concentration (THb) in a blood sample that could possibly contain as many as five different species of hemoglobin. Several different properties of whole blood have precluded such measurements. These characteristics include 1) the complex chemical mixture of as many as five individual hemoglobin species that are present in varying concentrations in a typical blood sample, and 2) the complex optical properties of whole blood that result from a combination of true optical absorbance by multiple light-absorbing compounds (including the hemoglobins, bilirubin, and other pigments), and the pronounced light-scattering ability of the red blood cells that are present in high concentrations.
Many prior methods have been devised to measure the total hemoglobin concentration in the complex chemical mixture of oxy-, carboxy-, met-, deoxy-, and sulfhemoglobin that comprise the total hemoglobin composition. For example, Loretz (U.S. Pat. No. 4,357,105) teaches a method whereby the hemoglobin is released from the red blood cells by the process of hemolysis, i.e. by disrupting the red blood cell membrane. Simultaneously, the various hemoglobin species are diluted by reagent solutions that convert them into a single chemical form, cyanmethemoglobin, the concentration of which is then measured spectrophotometrically by conventional apparatus.
FIG. 1 presents a graph of the extinction coefficients (indicative of optical absorbance) as a function of radiation wavelength for five individual hemoglobin species. Because the individual hemoglobin species differ significantly from each other in their optical absorbance spectra, no method that employs a single wavelength can measure the total hemoglobin concentration spectrophotometrically. Therefore, the chief advantage of the many prior methods that convert the sundry species into a single known species is that only one wavelength is needed for the subsequent spectrophotometric measurement of the resulting single species. The disadvantages of the above-mentioned chemical conversion methods are that 1) hemolysis and accurate dilutions are needed, 2) the reagents are often toxic, 3) chemical conversion is slow, 4) the red blood cells and the chemical nature of the whole blood sample are so drastically altered that the same sample cannot be used for further subsequent analyses of other blood constituents, and 5) the chemical conversion of the various hemoglobin derivatives into a single species makes it impossible to measure the original concentrations of the several individual species. The latter disadvantage is a serious limitation because the concentrations of oxy-, carboxy-, met-, deoxy-, and sulfhemoglobin provide valuable diagnostic information in many different medical applications.
In several patented methods (U.S. Pat. Nos. 4,134,678; 4,013,417; 3,972,614; and 4,997,769), the need for chemical conversion of the various hemoglobin derivatives into a single hemoglobin species has been eliminated by using multiple appropriate wavelengths to measure the concentration of each species. In such methods, the blood sample is either hemolyzed and diluted (Brown et al., U.S. Pat. No. 4,134,678; Raffaele, U.S. Pat. No. 4,013,417) or just hemolyzed (Johansen et al., U.S. Pat. No. 3,972,614; Lundsgaard, U.S. Pat. No. 4,997,769) to eliminate the light scattering by the red blood cells. The hemolyzed sample is then irradiated with at least one distinct wavelength for each of the three, four, or five hemoglobin species presumed to be present in the sample. By measuring the optical density of the hemolyzed sample with at least as many different wavelengths as there are unknown individual hemoglobin species, such methods yield a set of simultaneous equations that can subsequently be solved for the relative concentration of each of the various hemoglobin derivatives. These prior methods eliminate the need for chemically converting the various hemoglobin derivatives into a single species, but they suffer from the disadvantage of requiring a complex, bulky, and expensive apparatus comprised of pumps, plumbing, associated control circuitry, and in some cases, ultrasonic hemolyzers to dilute and hemolyze the blood sample. Finally, the techniques that employ hemolysis have two additional disadvantages: 1) their complicated plumbing systems are prone to clogging by blood residue, and 2) they aspirate and destroy the blood sample so that it cannot be retrieved and subjected to further analysis of other constituents.
In whole blood, the major obstacle to making optical measurements is the intense light scattering caused by the highly concentrated red blood cells, e.g. 5.4.times.10.sup.6 RBCs/.mu.l for human males. Consequently, all prior art has relied exclusively on hemolysis or on both hemolysis and dilution to reduce or eliminate the light scattering caused by red blood cells. This light scattering arises because the blood plasma and the red blood cells have different indices of refraction (B. Barer and S. Joseph, Quart. J. Microsc. Sci. 95:399,1954). Many uncontrollable factors make such light scattering unpredictable from one blood sample to another, including 1) the different plasma protein concentrations that determine the refractive index of plasma, 2) the aggregation and sedimentation of red blood cells in the sample, 3) the different hemoglobin concentrations inside the red blood cells that alter their refractive index, and 4) the size and shape of the red blood cells. The size and shape of the red blood cells differ from one animal species to another; they are also altered by disease states (e.g. sickle-cell anemia and spherocytosis); and they are altered by the osmotic pressure of the blood. Thus all prior methods which measure the total hemoglobin concentration and the relative concentrations of individual hemoglobin species have employed hemolysis to eliminate this intense, unpredictable light scattering caused by the red blood cells.
Prior art that relies on hemolysis to eliminate the intense light scattering by the red blood cells has the further disadvantage that even after thorough hemolysis the sample can be relatively turbid (L. R. Sehgal et al., Critical Care Medicine, 12:907-909, 1984). This residual turbidity in hemolyzed blood is small compared with the intense light scattering of red blood cells in unaltered whole blood (J. M. Steinke and A. P. Shepherd, IEEE Trans. Biomed. Eng. BME-33:294-301, 1986). Nevertheless, the residual turbidity in hemolyzed blood causes troublesome errors in the hemoglobin measurements. Thus, an additional disadvantage of prior apparatus that rely on hemolysis is the residual turbidity that results from a small number of unlysed red blood cells, lipid particles such as chylomicrons (a normal constituent of plasma that persists after hemolysis), light-scattering cell fragments produced by the hemolysis process, and other causes that are unknown. Although Lundsgaard (U.S. Pat. No. 4,997,769) teaches a method for dealing with the residual turbidity that remains after the hemolysis process, it would be advantageous to eliminate hemolysis completely and make spectrophotometric measurements directly in unaltered whole blood.
The Applicants have found that the scattering of light by unaltered (unhemolyzed) whole blood differs in five crucial ways from the turbidity of hemolyzed blood. First, as mentioned previously, the turbidity of hemolyzed blood is insignificant in magnitude in comparison with the light scattering of unhemolyzed whole blood. This conclusion is reinforced by FIG. 2 which shows a measure of light scattering in whole blood before and after hemolysis. For these data, the light scattering in unaltered, whole blood is approximately 20 times greater than after hemolysis. In fact, light scattering by red blood cells in unaltered whole blood is so intense that scattered light predominates over unscattered light after it has passed through as few as 10 layers of red blood cells (C. C. Johnson. J. Assoc. Adv. Med. Instrument. 4:22-27,1970). Prior art was not designed to accommodate the greater magnitude of the scattering effects of unaltered whole blood.
Second, a fundamental observation is that red blood cells scatter light at relatively large angles (J. M. Steinke and A. P. Shepherd, Applied Optics 27:4027-4033,1988). The Applicants have discovered that, even though most of the light is scattered at small angles, the magnitude of large-angle light scattering is sufficient to cause serious errors in spectrophotometric measurements on whole blood. Prior art does not address the problem of designing practical instruments by capturing the large-angle light scattering of unhemolyzed blood.
Third, the light scattering by red blood cells varies with wavelength in a complicated manner due to physical factors such as the relative size of red blood cells with respect to the wavelength, and the shape of the red blood cells. FIG. 3 shows the wavelength dependence of light scattering by RBCs as predicted by optical theory (R. N. Pittman and B. R. Duling, J. Appl. Physiol. 38:315-320,1975; R. N. Pittman, Ann. Biomed. Eng. 14:119-137,1986). Prior art does not address the complex wavelength dependence of the scattering effects of unaltered whole blood.
Fourth, as FIG. 4 shows, the light scattering by red blood cells is not only a function of wavelength, but it also depends on the composition of the hemoglobin contained in the red blood cells, i.e. the light scattering depends on the relative concentrations of oxy-, deoxy-, carboxy-, met-, and sulfhemoglobin in the red blood cells. Prior art does not address the complex dependence of light scattering on the concentrations of the individual hemoglobin species in unaltered whole blood.
Fifth, many poorly understood, uncontrolled processes occur in unaltered, whole blood that change its optical properties. Such miscellaneous causes of unpredictable light scattering include 1) the different plasma protein concentrations that determine the refractive index of plasma in one sample versus another, 2) the aggregation of red blood cells in the sample, 3) the different hemoglobin concentrations inside the red blood cells that alter their refractive index, 4) the size and shape of the red blood cells, 5) chylomicrons or other light-scattering lipid particles, 6) cell fragments, 7) microscopic clots, and 8) light-sieving effects of sedimented RBCs.
As shown in the comparative example below, the apparatus of Lundsgaard, U.S. Pat. No. 4,997,769, fails to yield valid results when measuring a sample of unaltered (unhemolyzed) whole blood. Similarly, other prior art that relies on hemolysis such as Brown et al., U.S. Pat. No. 4,134,678; Raffaele, U.S. Pat. No. 4,013,417; and Johansen et al., U.S. Pat. No. 3,972,614 would also fail to yield valid results on unhemolyzed whole blood because these methods also fail to capture the light scattered at large angles by the sample, and because their measurements do not take into account the magnitude, the wavelength dependence, or the hemoglobin-composition-dependence of the light-scattering effects of unaltered whole blood.